CN104450637A - Fusion protein CR2-Linker-GDH and application thereof - Google Patents
Fusion protein CR2-Linker-GDH and application thereof Download PDFInfo
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Abstract
The invention discloses a fusion protein CR2-Linker-GDH and the application of the fusion protein CR2-Linker-GDH, and belongs to the technical field of genetic engineering and biological catalysis. Fusion expression is carried out on carbonyl reductase and glucose dehydrogenase to achieve coupling between the two enzymes, and a separated and purified fusion protein in a successfully constructed recombinant bacteria package is utilized to carry out biotransformation COBE to obtain S-CHBE. By optimizing reaction conditions, a substrate of the fusion protein can be totally converted, the optical purity of a product is above 99%, and the TTN reaches up to 1200. The final concentration of the substrate is 305 mmol/L through a substrate batch replenishing strategy, the yield of the product reaches up to 53%, the product of 162 mmol/L is obtained, and the e.e. value remains above 99%. The fusion protein CR2-Linker-GDH and the application of the fusion protein CR2-Linker-GDH provide a novel research thought for coupling a coenzyme regeneration cycle approach into a biotransformation approach of the S-CHBE and have great significance for simplifying the a chiral transformed way.
Description
Technical field
The present invention relates to a kind of fusion rotein CR2-Linker-GDH and application thereof, belong to genetically engineered and biocatalysis technology field.
Background technology
Chiral alcohol compounds is the important intermediate in the fields such as class medicine/food/chemistry of pesticide/fine chemistry industry/makeup.Optically pure (S)-4-chloro-3-hydroxyl ethyl butyrate [(S)-CHBE] is the precursor substance of synthesis anticholesteremic agent atorvastatin (Atovastatin)/medicine such as Hydroxymethylglutaryl methylacyl coenzyme A (HMG-CoA) reductase inhibitor and alkaloid SlageninsB and C.In its production method, biological process has the advantages such as reaction conditions gentleness/reactions steps is few, and the method has very strong solid and enantioselectivity.
The theoretical yield being carried out bio-transformation by oxydo-reductase can reach 100%, but in the application of oxydo-reductase, except needing suitable enzyme, also needs the regenerating coenzyme system providing efficient/low cost.The biological process regeneration of coenzyme generally includes enzyme and is coupled method and substrate is coupled method, and wherein enzyme is coupled method is utilize two parallel redox reaction enzyme systems, and a substrate for enzymatic activity transforms, and another enzyme is catalysis coenzyme cyclic regeneration then.
Be coupled in method regeneration system at traditional enzyme, need to add substrate katalaze enzyme and coenzyme cyclophorase two kinds of enzymes respectively, troublesome poeration, cost is high, and coenzyme is two intermolecular transmission of enzyme, likely causes regenerating coenzyme efficiency not high.Utilizing convergence strategy to build artificial enzyme likely makes the avtive spot of two enzymes close, thus improves catalytic efficiency.
Phosphorous acid desaturase (PTDH) can realize the regeneration of NADPH using the phosphite of cheapness as substrate, Torres
etc. the fusion enzyme constructing a series of B-V monooxygenase (BVMOs) and PTDH, when NADP concentration only has 5umol/L, still can realize the effective conversion (79%, 3h) of Propiophenone to jasmal, after merging, each enzyme can keep the catalytic activity of natural enzyme.The fusion enzyme that H lsch etc. constructs hydrogenlyase (FDH) and 3-ketone acyl-acp reductase enzyme (KR) carries out chiral alcohol production; compared with two enzyme mixtures; initial catalyst speed improves 2 times; substrate conversion efficiency can reach 99.97%, and has high optical selective (99.9% (S)-1-(pentafluorophenyl) ethanol).
The present invention constructs a kind of fusion rotein, possesses the activity of carbonyl reductase and Hexose phosphate dehydrogenase simultaneously, with certain density 4-chloroacetyl acetacetic ester (COBE) for substrate, glucose is cosubstrate, utilize fusion rotein to carry out bioconversion reaction, achieve the dual-use function of catalysis and coenzyme circulation.
Summary of the invention
The object of this invention is to provide a kind of fusion rotein, possess the activity of carbonyl reductase and Hexose phosphate dehydrogenase simultaneously, the present invention utilizes genetic engineering technique to realize the fusion of two kinds of zymoproteins, achieves the Successful integration of two enzyme function.Utilize this fusion rotein for catalyzer can asymmetric transformation preparation (S)-4-chloro-3-hydroxyl ethyl butyrate.
The aminoacid sequence of described carbonyl reductase is the sequence shown in SEQ ID NO.1.
The aminoacid sequence of described Hexose phosphate dehydrogenase is the sequence shown in SEQ ID NO.2.
Encode between described carbonyl reductase and the aminoacid sequence of Hexose phosphate dehydrogenase and be connected with oligopeptides joint: (GGGGS)
3.
In one embodiment of the invention, described oligopeptides joint is flexible oligopeptides joint (GGGGS)
3.
The aminoacid sequence of described fusion rotein is the sequence shown in SEQ ID NO.3.
The nucleotide sequence of the gene of encoding said fusion protein is the sequence shown in SEQ ID NO.4.
The present invention also provides a kind of recombination bacillus coli of expressing described fusion rotein.
The present invention also provides the application of a kind of described fusion rotein in preparation (S)-4-chloro-3-hydroxyl ethyl butyrate.
Described application, in one embodiment of the invention, be take COBE as substrate, glucose is cosubstrate, and fusion rotein is catalyzer, adds quantitative NADP
+, carry out asymmetric transformation reaction, obtain (S)-4-chloro-3-hydroxyl ethyl butyrate.
Described application, in one embodiment of the invention, is take COBE as substrate, at 30 DEG C, in the acetate buffer solution of pH 5.5, adds 7U fusion rotein CR2,0.05mmol/LNADP
+, reaction 90min.This embodiment is the embodiment of Optimal reaction conditions, preferably descends productive rate to be 100%.
The present invention, by the encoding gene amalgamation and expression by carbonyl reductase and Hexose phosphate dehydrogenase two enzymes, realizes the coupling of these two enzymes, and the fusion protein expression of the recombinant bacterium that success builds can reach 0.143g/g (albumen/thalline).The Km of the carbonyl reductase of fusion rotein of the present invention is 1.4 times that the single carbonyl reductase Km merged does not occur, and is that 58.82 μm of ol/min*mg improve 16% than the Vmax value 50.66 of single albumen, catalytic efficiency k to the Vmax of substrate COBE
cat/ Km is 2.8 times of single PROTEIN C R2 catalytic efficiency.
The present invention also utilizes the fusion rotein in born of the same parents after separation and purification to carry out bio-transformation COBE and obtains S-CHBE, and substrate can transform completely, the optical purity >99% of product, and complete turnover number (TTN) is up to 1200.The optimal pH of fusion rotein catalyzed conversion COBE is 5.5, and optimum temperuture is 30 DEG C of conditions, the suitableeest coenzyme concentration 0.05mmol/L, reaction conversion ratio 100%, the e.e. value >99% under optimum condition.By substrate feeding strategy in batches, Final substrate concentrations is 305mmol/L, and the yield of product, up to 53%, obtains the product of 162mmol/L, and e.e. value keeps >99%.The biotransformation pathway that these work are coupled into S-CHBE for regenerating coenzyme circulation approach provides new Research Thinking, significant for simplification chiral inversion approach.The present invention is the biological function of Successful integration two kinds of oxydo-reductase, and efficient economy prepares chipal compounds provides a brand-new thinking.
Embodiment
The detection of product optical purity: adopt HPLC to analyze, chromatographic column is Chiralcel OB-H post (4.6mm × 25cm; DaicelChemical Ltd; Japan), moving phase is normal hexane/Virahol (9:1), and flow velocity is that the appearance time of 0.5mL/min, S-CHBE and R-CHBE is respectively 19.4min and 16.5min.Product e.e. value is obtained according to calculated by peak area.
Substrate/production concentration is quantitative: adopt GC to analyze, chromatographic column is Varian PEG-20M capillary column, vaporizer temperature 250 DEG C, detector temperature 300 DEG C, and column temperature retains 2.5min from 60 DEG C, is warming up to 180 DEG C, then keeps 6.5min with 20 DEG C/min.Inside be designated as benzene pentanone (Valerophenone).Benzene pentanone, CHBE appearance time is respectively 11.2min, 11.5min.
The calculating of product (S)-4-chloro-3-hydroxyl ethyl butyrate mapping excessive value:
Mapping excessive value (e.e.%)=[(CS-CR)/(CS+CR)] * 100%
The calculating of product (S)-4-chloro-3-hydroxyl ethyl butyrate productive rate: productive rate (%)=CS/C0*100%
Wherein, in formula, CS is the concentration of (S)-enantiomorph after reaction, and CR is the concentration of (R)-enantiomorph after reaction, and C0 is the concentration of substrate 4-chloroacetyl acetacetic ester before reaction.
Embodiment 1: the acquisition of fusion gene
Adopt the method by extracting plasmid, pcr amplification from E.coliBL21/pET-CR2 and E.coliBL21/pET-GDH, or the method for directly chemosynthesis, obtain the nucleotide sequence (respectively as shown in SEQ ID NO.5, SEQ ID NO.6) of carbonyl reductase CR2 and Hexose phosphate dehydrogenase GDH, its aminoacid sequence is respectively as shown in SEQ ID NO.1, SEQ ID NO.2.
Adopt the method for Overlap extension PCR, the oligopeptides joint (GGGGS) that interpolation one is flexible between two genes
3, obtain amalgamation and expression gene cr2-linker-gdh.The nucleotide sequence of fusion gene is as shown in SEQ ID NO.4, and the aminoacid sequence of its coding is as shown in SEQ ID NO.3.
The preparation method of described fusion gene, specifically:
(1) with plasmid pET-cr2 for template, utilize upstream primer 1 (sequence is as shown in SEQ ID NO.7), downstream primer 2 (sequence is as shown in SEQ ID NO.8), by PCR reaction amplification cr2 gene fragment;
(2) with plasmid pET-gdh for template, utilize upstream primer 3 (sequence is as shown in SEQ ID NO.9), downstream primer 4 (sequence is as shown in SEQ ID NO.10), by PCR reaction amplification gdh gene fragment.
(3) cr2, gdh two fragment after overlapping connection of annealing in 72 DEG C of effect downward-extensions at archaeal dna polymerase to complete double-strand; With the complete double-stranded DNA of last round of acquisition for template, primer 1 and primer 4PCR is utilized to obtain amalgamation and expression gene cr2-linker-gdh.
Embodiment 2: the structure of the genetic engineering bacterium of expressed fusion protein CR2-Linker-GDH
(1) be connected on pMD-19T carrier after the fusion gene that embodiment 1 obtains being carried out purifying, transformation of E. coli E.coliJM109 competent cell, screen correct recombinant plasmid pMD-C-L-G, and sequence verification;
(2) utilize restriction enzyme Nde I and Not I to carry out enzyme to recombinant plasmid pMD-C-L-G and pET-28a respectively to cut, glue reclaims and purifying digestion products, linearizing pET-28a carrier is connected under the effect of T4 ligase enzyme with c-l-g gene fragment, obtain the recombinant plasmid with two goal gene cr2 and gdh, screen correct recombinant plasmid, called after pET-C-L-G.
(3) by recombinant plasmid pET-C-L-G transformed competence colibacillus E.coliBL21 (DE3) obtained in the previous step, final positive strain E.coli BL21/pET-C-L-G is obtained.
Embodiment 3: the abduction delivering of fusion rotein
The mono-colony inoculation of picking recombinant bacterial strain E.coli BL21/pET-C-L-G contains in the LB liquid nutrient medium of the kantlex of 50 μ g/mL in 5mL, in 37 DEG C, 200rpm shaking culture is spent the night, get 1mL nutrient solution to transfer in 50ml containing in the LB liquid nutrient medium of 50 μ g/mL kantlex, in 37 DEG C, after 200rpm shaking culture is 0.6-0.8 to OD, in culture, add inductor sec.-propyl-D-thiogalactoside IPTG 0 ~ 1mmol/L, inducing culture 10h at 17 DEG C; The centrifugal 10min of 10,000rpm, collects thalline, with brine twice, collects and obtains the full cell of recombinant bacterium.Fusion protein expression can reach 0.143g/g (albumen/thalline)
Complete for the recombinant bacterium of collection cell is resuspended in the sample introduction damping fluid of purifying, ultrasonication: working hour 2s, intermittent time 3s, altogether 20min; The centrifugal 30min of broken liquid 12000rpm; Abandon precipitation, get supernatant and carry out follow-up protein purification work;
Protein purification: the Ni post first supernatant being hung QIAGEN company, with the elution buffer wash-out foreign protein of 10-15%, with the elution buffer wash-out target protein of 20%, finally use the elution buffer wash-out foreign protein of 30%, collect target protein and carry out ultrafiltration and concentration and desalination.More than 95% is reached through SDS-PAGE testing goal purity of protein.
Embodiment 4: the application of fusion rotein CR2-Linker-GDH
Take COBE as substrate, glucose is cosubstrate, and fusion rotein is catalyzer, adds quantitative NADP
+carry out asymmetric transformation reaction: in the acetate buffer solution of 1mL 0.1mol/L pH4.0-6.0, or in the phosphoric acid buffer of 1mL 0.1mol/L pH6.0-8.0, or in the Tris-HCl damping fluid of 1mL 0.1mol/L pH8.0-9.0, the concentration of substrate 4-chloroacetyl acetacetic ester is 10g/L, glucose concn 13.5g/L, NADP+ concentration 0.012 ~ 0.2mmol/L, temperature of reaction is 25-50 DEG C.After reaction terminates, get 100 μ L reaction solutions, add mark benzene pentanone in 10 μ L, 90 μ L extraction into ethyl acetate, organic phase is analyzed.Get 500 μ L reaction solutions, add 500 μ L n-hexane extractions, organic phase is analyzed.
Embodiment 5: different condition is on the impact of catalyzed reaction
Utilize the target protein catalyzed conversion COBE after purifying, explore different condition to the impact of changing effect.Take COBE as substrate, glucose is cosubstrate, and fusion rotein is catalyzer, adds quantitative NADP+, carries out asymmetric transformation reaction.
(1) pH is reacted on the impact of fusion rotein system catalytic asymmetric reduction COBE
1mL reaction system, COBE is 10g/L, glucose is 13.5g/L, coenzyme 0.02mmol/L, in different pH damping fluid (4.0-9.8), the acetate buffer solution of 0.1mol/L pH4.0-6.0, or the phosphoric acid buffer of 0.1mol/L pH6.0-8.0, or in the Tris-HCl damping fluid of 0.1mol/L pH8.0-9.0, react under 35 DEG C of conditions.Get 100 μ L reaction systems, add mark in 10 μ L, 90 μ L extraction into ethyl acetate, air inlet phase.Found that, fusion rotein is best in pH 5.5 times catalyzed conversion COBE effects.
(2) temperature of reaction is on the impact of fusion rotein system catalytic asymmetric reduction COBE
Get coenzyme concentration 0.02mmol/L when receiving maximum TTN value, 1ml reaction system, COBE is 10g/L, and glucose is 13.5g/L.React at condition of different temperatures 25,30,35,40,45,50 DEG C, get 100 μ L reaction systems, add mark in 10 μ l, 90 μ L extraction into ethyl acetate, air inlet phase.Found that, fusion rotein catalyzed conversion COBE effect at 30 DEG C is best.
(3) enzyme catalyst concentration and coenzyme concentration are on the impact of fusion rotein catalyzed reaction efficiency
In 1mL reaction system, COBE is 10g/L, and glucose is 13.5g/L, pH of buffer 5.5, under 30 DEG C of conditions, scope 0.012 ~ the 0.2mmol/L of coenzyme, the scope CR22 ~ 8U of fusion rotein, reaction 90min, obtain the suitableeest coenzyme concentration 0.05mmol/L, catalyst concn 7U, reaction conversion ratio 100%, e.e. value >99%.
By condition optimizing, reaction optimum condition is: 30 DEG C, reaction system pH 5.5, coenzyme concentration 0.05mmol/L.
Embodiment 6 zymologic property or other
(1) fusion rotein C-L-G optimal pH in enzymatic reaction and pH stability
In fusion rotein, the optimal pH of carbonyl reductase and Hexose phosphate dehydrogenase and stability are separately surveyed.
Carbonyl reductase: cumulative volume is that the system of 100 μ L comprises 20mmol/L COBE, 1.2mmol/L NADPH and 0.1mol/L different pH buffer, at 30 DEG C of constant temperature 2min, adds appropriate enzyme liquid and measures the work of its enzyme, investigate the optimal pH of this enzyme.Using the highest enzyme work in mensuration different pH damping fluid to live contrast as 100% enzyme, the lower institute of other pH condition surveys enzyme work and represents with the per-cent contrasting enzyme work.
By electrophoretically pure enzyme liquid in different pH damping fluid, after placing 24h respectively in 4 DEG C, the enzyme liquid getting equivalent measures remnant enzyme activity, investigates the pH stability of this enzyme.With 4 DEG C, the enzyme work of the pure enzyme liquid of the fresh electrophoresis under pH 5.5 condition is lived as 100% enzyme and is contrasted, and under other pH condition, enzyme work represents with the per-cent contrasting enzyme alive.
Hexose phosphate dehydrogenase: cumulative volume is that the system of 100 μ L comprises 10mmol/L glucose, 1.2mmol/LNADPH and 0.1mol/L different pH buffer, and other conditions are the same.
Found that, the CR2 of fusion rotein is more stable under acidity and neutrallty condition, and GDH is more stable under pH 5.5-6.5 and solutions of weak acidity.
(2) fusion rotein C-L-G enzymatic reaction optimum temperuture and temperature stability
In fusion rotein, the optimum temperuture of carbonyl reductase and Hexose phosphate dehydrogenase and temperature stability are separately surveyed.
Carbonyl reductase: cumulative volume is that the system of 100uL comprises 20mmol/L substrate COBE, 1.2mmol/L coenzyme NADP 11 and 0.1mol/L sodium-acetate (pH 5.5) damping fluid, constant temperature 2min in the water-bath of differing temps (25 ~ 70 DEG C), add the pure enzyme liquid of appropriate electrophoresis respectively and measure the work of its enzyme, investigate the optimum temperuture of this enzyme.With enzyme work the highest at surveyed differential responses temperature be 100% enzyme live (contrast), other temperature condition lower survey enzyme work with contrast enzyme work per-cent represent.
Pure for electrophoresis enzyme liquid is incubated 12h respectively under differing temps (25 ~ 70 DEG C), and the enzyme liquid then getting equivalent measures its remnant enzyme activity, investigates the thermostability of this enzyme.Under pH 5.5 condition, the enzyme work of the pure enzyme liquid of fresh electrophoresis is lived as 100% enzyme and is contrasted, and at other temperature, enzyme work represents with the per-cent contrasting enzyme alive.
Hexose phosphate dehydrogenase: cumulative volume is that the system of 100 μ l comprises 10mmol/L glucose, 1.2mmol/L NADP
+with 0.1mol/L sodium-acetate (pH 5.5) damping fluid, other conditions are the same.
The optimal reactive temperature drawing the CR2 catalytic reduction COBE of fusion rotein is by experiment 50 DEG C, and the optimal reactive temperature of GDH oxidizing glucose is 40 DEG C.The CR2 of fusion rotein has and is greater than 70% enzyme activity between 35-52 DEG C, and GDH has and is greater than 70% enzyme activity between 30-42 DEG C.Result shows simultaneously, and fusion rotein CR2 all can keep the vigor of more than 50% between 25 ~ 40 DEG C.
(3) metal ion affects fusion rotein C-L-G enzymatic reaction
Choose Mg
2+, Zn
2+, Ca
2+, Mn
2+, Ni
2+, Cu
2+, Co
2+, Fe
3+and EDTA, investigate the impact that it is lived on fusion protease.
Found that EDTA can suppress the activity of fusion rotein, record enzyme under conditions and live as not adding 28% during EDTA.Illustrate that fusion rotein shows as metal ion dependency.Fe
3+suppress catalytic activity completely, all the other divalent-metal ions all can improve its activity, are wherein adding the Ca of 25mM
2+time enzyme live improve maximum, reach 412% of contrast.
(4) CR2 enzyme kinetics character research in fusion rotein C-L-G
Fusion rotein CR2 Determination of Kinetic Parameters: be variable (0.3 ~ 1.2mmol/L) respectively with coenzyme NADP 11, under different substrate COBE concentration, measure enzyme activity, investigate different substrate COBE concentration (0.5 ~ 5mmol/L) to the impact of CR2 enzyme ' s reaction speeding.
Calculate through experiment mapping, obtain the kinetic property parameter of fusion rotein CR2, Michaelis-Menton constant Km is the constant characterizing avidity between enzyme-to-substrate, and Km is less, represent that the avidity between enzyme-to-substrate is larger, the coenzyme amount namely reached required for maximum speed of reaction one half is less.The Km (1.17mmol/L) of fusion rotein CR2 is 1.4 times of its Km of single PROTEIN C R2 (0.83mmol/L), can infer, in fusion rotein CR2 reaction system, larger compared to the demand of single PROTEIN C R2 to reduced coenzyme NADPH.Fusion rotein CR2 is 58.82umol/min*mg to the Vmax of substrate COBE, and the Vmax value 50.66 of more single PROTEIN C R2 is bigger than normal.Fusion rotein CR2 catalytic efficiency k
cat/ Km is 1.0*10
5l/mol*S is 2.8 times of single PROTEIN C R2 catalytic efficiency.
Although the present invention with preferred embodiment openly as above; but it is also not used to limit the present invention, any person skilled in the art, without departing from the spirit and scope of the present invention; all can do various changes and modification, what therefore protection scope of the present invention should define with claims is as the criterion.
Claims (10)
1. a fusion rotein, is characterized in that, described fusion rotein comprises carbonyl reductase and Hexose phosphate dehydrogenase.
2. fusion rotein according to claim 1, is characterized in that, the aminoacid sequence of described carbonyl reductase is the sequence shown in SEQ IDNO.1; The aminoacid sequence of described Hexose phosphate dehydrogenase is the sequence shown in SEQ ID NO.2.
3. fusion rotein according to claim 1, is characterized in that, encodes between described carbonyl reductase and the aminoacid sequence of Hexose phosphate dehydrogenase and is connected with oligopeptides joint (GGGGS)
3.
4. fusion rotein according to claim 1, is characterized in that, the aminoacid sequence of described fusion rotein is the sequence shown in SEQ ID NO.3.
5. fusion rotein according to claim 1, is characterized in that, the nucleotide sequence of encoding said fusion protein is the sequence shown in SEQ IDNO.4.
6. carry the carrier of the gene of the arbitrary described fusion rotein of coding claim 1-5.
7. express the genetic engineering bacterium of the arbitrary described fusion rotein of claim 1-5.
8. the application of the arbitrary described fusion rotein of claim 1-5 in preparation (S)-4-chloro-3-hydroxyl ethyl butyrate.
9. application according to claim 8, is characterized in that, described application take COBE as substrate, and glucose is cosubstrate, and fusion rotein is catalyzer, adds quantitative NADP
+, carry out asymmetric transformation reaction, prepare (S)-4-chloro-3-hydroxyl ethyl butyrate.
10. application according to claim 9, is characterized in that, described application take COBE as substrate, at 30 DEG C, in the acetate buffer solution of pH 5.5, adds 7U fusion rotein CR2,0.05mmol/L NADP
+, reaction 90min.
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| CN106754776A (en) * | 2016-12-26 | 2017-05-31 | 江南大学 | The GDH mutant that a kind of specific enzyme activity for being catalyzed xylose is improved |
| CN109326326A (en) * | 2018-09-28 | 2019-02-12 | 青岛农业大学 | A kind of separation and application of rainbow conk xyloside transferase |
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| CN112941124A (en) * | 2021-02-09 | 2021-06-11 | 江苏阿尔法药业股份有限公司 | Method for preparing eligerstrode intermediate through whole-cell catalysis |
| CN113652407A (en) * | 2021-07-09 | 2021-11-16 | 浙江工业大学 | Carbonyl reductase mutant and application thereof in asymmetric synthesis of bi-chiral compound |
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